Thyrotropin Stimulation of the Lutropin/Choriogonadotropin Receptor: Different Sites Mediate Agonist Activity and High Affinity Binding

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Abstract
Surprisingly, thyrotropin (TSH) can increase cAMP and inositol phosphate (IP) levels in Cos-7 cells transfected with the lutropin (LH)/choriogonadotropin (CG) receptor (LH/CGR) as well as LH or CG, as evidenced by similar EC50 and maximal stimulation values. Additionally surprising, TSH activation is evident, despite markedly reduced levels of high affinity TSH binding by comparison to CG (Hidaka A, et al. 1993 Biochem Biophys Res Commun 196:187-195). In this report, we questioned whether the unusual TSH activity, as well as the discrepancy between TSH activity and binding, might reflect the existence of distinct agonist and binding sites on the LH/CGR extracellular domain and the ability of TSH to interact with the former despite a minimal interaction with the latter. We evaluated this possibility by using two chimeras spanning the extracellular domain of the TSHR and the LH/CGR: Mcl + 2, where residues 8-165 of the TSHR are substituted, and Mc2 + 3 + 4, where residues 90-370 are replaced with the corresponding peptide segment from the LH/CGR. After transfection in Cos-7 cells, Mc2 + 3 + 4 exhibits higher affinity for CG than wild-type LH/CGR, but has no CG agonist response in assays measuring cAMP or inositol phosphate (IP) levels. Conversely, the Mcl + 2 chimera exhibits significantly decreased affinity for CG, but CG agonist activity is comparable to wild-type LH/CGR in cAMP and IP assays. These data show that the extracellular domain of the LH/CGR does have distinct sites for CG binding and agonist activity: the C-terminus in Mc2 + 3 + 4 is important for high affinity CG binding, whereas the N-terminus in Mcl + 2 is able to exhibit a CG agonist response, despite low affinity binding. When evaluated using TSH, Mcl + 2, with the C-terminus of the TSHR present, exhibits high affinity TSH binding comparable to wild-type TSHR. Unexpectedly, Mcl + 2, with the substitution of the N-terminus of the extracellular domain of the LH/CGR, exhibits even better TSH agonist activity than wild-type TSHR, not a loss of activity. Thus, the N-terminus of the extracellular domain of the LH/CGR can couple TSH binding to signal transduction events even better than the N-terminus of the TSHR. This may, in part, explain why TSH has an unusual agonist activity in cells transfected with LH/CGR, despite relatively low affinity binding. Although distinct agonist and binding sites exist in the linear sequence of the extracellular domain, the activity of the two sites is interdependent. Thus, Mcl + 2 retains the linear epitopes for reactivity with blocking TSHR autoantibodies; however, the ability of these antibodies to inhibit TSH-increased cAMP levels is reduced by comparison to wild-type TSHR. Three of the four blocking TSHR IgGs were, in addition, able to inhibit hCG-increased cAMP levels in Mcl + 2-transfected cells better than inhibition of TSH-stimulated cAMP levels in the same cells. There is, therefore, an altered interaction between the N- and C-terminus in Mcl + 2; as a result, high affinity TSH binding overcomes the blocking TSHRAb inhibition, whereas low affinity hCG binding does not and inhibition persists.